A reliable, quantitative and non-invasive method for the characterization of the molecular changes associated with early cataractogenesis in vivo has long been an important goal of human clinical cataract research. Such a method would allow researchers and physicians to (a) assess the effectiveness of putative anticataract reagents; (b) evaluate the cataractogenic role of pharmacologic agents or radiation used in the treatment of systematic diseases; (c) characterize early cataract in epidemiologic studies of human or animal populations subject to differential cataractogenic stress; and (d) provide a quantitative basis for the medical decision to intervene surgically or pharmaceutically in the treatment of cataract.
In 1975, T. Tanaka and G. Benedek ("Observation of Protein Diffusivity in Intact Human and Bovine Lenses with Application to Cataract," Invest. Opthal. 14:449-456, 1985) showed that the Brownian motion of proteins in excised human and bovine lenses could be measured optically using the method of quasielastic light scattering (QLS) spectroscopy. Following this work, T. Tanaka and C. Ishimoto ("In Vivo Observation of Lens Protein Diffusivity in Normal and X-Irradiated Rabbit Lenses," Exp. Eye Res. 39:61-68, 1984) demonstrated that QLS could be used in vivo in rabbits to detect changes in mean protein diffusivity as a function of age and position in the rabbit ocular lens. Further observations showed that the cataractogenic insult of x-irradiation upon the rabbit lens produced dramatic changes in the form of the autocorrelation function of the scattered light at a very early stage in the cataractogenic process. The autocorrelation function is an important tool for mathematical analysis of QLS. This change in the autocorrelation function demonstrated that the x-irradiation was responsible for drastic changes in the diffusivity of the protein scattering elements undergoing Brownian movement within the ocular tissue. Both Nishio and the 1977 Tanaka team observed that these altered correlation functions had a form different from that expected for the Brownian motions of a single-type scatterer. However, neither understood a quantitative analysis of the information contained in the non-exponential character of the autocorrelation function observed.
In 1986, T. Libondi et al. ("In Vivo Measurement of the Aging Rabbit Lens Using Quasielastic Light Gathering," Curr. Eye. Res. 5(6):411-419, 1986) showed that the form of the autocorrelation function of the scattered light from a living rabbit eye indicated the presence of at least two distinct diffusing aspects within the rabbit lens. One species had a diffusivity corresponding to the .alpha.-crystalline protein. The other was a much more slowly diffusing species of the type discovered in vitro by M. Delaye et al. ("Identification of the Scattering Elements Responsible for Lens Opacification in Cold Cataracts," Biophys. J. 37:647-656, 1982).
In recent years QLS has been used to study the ocular lens in vivo and in vitro. A method and apparatus for analyzing QLS is described in U.S. Pat. No. 4,957,113, and U.S. Pat. No. 5,072,731, respectively, which are incorporated herein by reference.
The techniques described in the above-reference patents are capable of quantitating the amount of light scattered by diffusing chemical species in a medium, as well as their rates of diffusion. With QLS, the temporal fluctuations in intensity of light scattered by a selected small volume in the lens which is illuminated by an incident laser beam are studied. The scattered light intensity fluctuates in time because of the Brownian motion of the scattering elements. Brownian motion is defined as the motion of macromolecules caused by thermal agitation and the random striking by neighboring molecules in a solution. In the lens of the human eye, the Brownian of protein molecules may be recorded and analyzed by quasielastic light scattering.
Research has shown that the principal scattering elements within the lens are the molecular constituents of the fiber cells. These constituents are principally globular proteins called crystallins. The aggregation of small proteins within the lens is the very first stage in the process of cataractogenesis. As the light scattering becomes more pronounced, it becomes noticeable to the clinician and is termed a cataract. However, this represents a late stage of a continuous process of increase in light scattering with time within the lens. By using information obtained from the light scattered by the various fast and slow moving protein species, it is possible to determine the degree of aggregation and thus the degree of cataractogenesis before it would be noted clinically.
The intensity fluctuations of the scattered light are detected by collecting the light scattered from the illuminated volume in the eye lens and focusing this light onto the surface of an optical square law detector such as a photomultiplier tube or solid-state photodiode. The output of the detector is a photoelectric current whose temporal fluctuations are synchronized with the fluctuations in the scattered light intensity. The temporal fluctuations in the photoelectric current can be mathematically analyzed to provide a quantitative measure of the degree of cataractogenesis.
The experimental data is typically expressed in the form of the temporal autocorrelation function, C(.tau.), of the intensity of the detected scattered light from the medium as a function of the delay time, .tau.. From the mathematical form of the autocorrelation function of the photoelectric current, it is possible to determine the diffusivity of the scattering elements undergoing Brownian movement. The decoded information has been shown clinically to provide an accurate quantitative measure of the source of increased light scattering on a molecular level long before cataract formation could be detected visually by either the subject or the physician.
The QLS inventions described in the above-referenced patents have provided tools to detect cataract formation at a very early stage. However, it has been determined that in situations where the lens contains a significant amount of immobile proteins, these methods do not produce results that are as accurate as desired. Therefore, it can be appreciated that there is a significant need for a method and apparatus that can produce results having the desired accuracy even in a lens containing immobile proteins. The present invention fulfills this need and provides other related advantages.